Defined cell culturing surfaces and methods of use

ABSTRACT

In one aspect, there is provided a cell culturing substrate including: 
     a cell culture surface having a film attached thereto, wherein the film includes one or more plasma polymerized monomers; and a coating on the film-coated surface, the coating deposited from a coating solution comprising one or more extracellular matrix proteins and an aqueous solvent, where the total extracellular matrix protein concentration in the coating solution is about 1 ng/mL to about 1 mg/mL.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a division of U.S. application Ser. No. 12/508,661,filed Jul. 24, 2009, now U.S. Pat. No. 8,288,513, which claims priorityto U.S. Provisional Patent Application No. 61/083,570, filed Jul. 25,2008, and to U.S. Provisional Patent Application No. 61/085,044, filedAug. 13, 2008, the entire contents of these applications beingincorporated by reference herein in their entireties.

FIELD OF THE INVENTION

This invention relates to defined surfaces for culturing cells. Moreparticularly, the present invention provides methods and materials forculturing embryonic stem cells and other adult stem cells on definedcell culture surfaces.

BACKGROUND OF THE INVENTION

Human embryonic stem (hES) cells typically require a substrate andculture medium to maintain indefinite self-renewal and pluripotencycharacteristics. The most common substrates for culturing hES cells aremonolayers of inactivated fibroblast feeder cells grown on tissueculture (TC) polystyrene surface or TC culturing vessels coated with anextracellular matrix (ECM), for example BD Matrigel™-coated TC culturingvessels. Both of these substrates are poorly defined and introduce ahigh degree of experimental variability. Since hES cells are thought tohave a significant potential implication in furthering knowledge ofdevelopmental biology, drug discovery and may play an important role infuture clinical applications, it is important to identify conditions forculturing these cells on defined surfaces.

SUMMARY OF THE INVENTION

In one aspect, there is provided a cell culturing substrate including: acell culture surface having a film attached thereto, wherein the filmincludes one or more plasma polymerized monomers; and a coating on thefilm-coated surface, the coating deposited from a coating solutioncomprising one or more extracellular matrix proteins and an aqueoussolvent, where the total extracellular matrix protein concentration inthe coating solution is about 1 ng/mL to about 1 mg/mL.

In other aspects, there is provided a method of preparing a cellculturing substrate including: providing a cell culture surface; plasmapolymerizing a film onto the surface to form a film-coated surface,wherein the plasma polymerizing utilizes one or more monomers; andintroducing a coating solution to the film-coated surface to form a cellculture substrate, the coating solution including one or moreextracellular matrix proteins and an aqueous solution, wherein the totalextracellular matrix protein concentration in the coating solution isabout 1 ng/mL to about 1 mg/mL.

In aspects, there is provided a method of culturing stem cellsincluding: providing a cell culturing substrate; applying a suspensionof stem cells to the cell culturing substrate; incubating the suspensionof stem cells on the cell culturing substrate at 5% CO₂ in humidifiedair at 37° C.; and permitting the stem cells to attach to the cellculturing substrate, wherein the attached cells remain in apredominately undifferentiated state. Unless differentiation is pinducedusing specific differentiation factors in media (E.G. EXAMPLE 10)

Advantageously, with subject to the invention, a cell culturingsubstrate may be provided that has good attachment characteristics forstem cells with favorable avoidance of stem cell differentiation.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts comparative colony attachment of crystal violet stainedhuman embryonic stem (hES) cells seeded on various substrates: tissueculture (TC)-treated polystyrene plates coated with extracellularmatrix, more specifically, Matrigel™, a complex mixture of extracellularmatrix proteins (FIG. 1A), TC treated polystyrene plates coated withhuman fibronectin (FIG. 1B); and plasma polymerized plates coated withhuman fibronectin (FIG. 1C) and growth media.

FIG. 2 comparison of typical hES cell colony morphology on selectedsubstrates: TC plates coated with an extracellular matrix, (such as,Matrigel™) (FIG. 2A) and plasma polymerized plates coated with humanfibronectin for 18 passages (FIG. 2B).

FIG. 3 Immunocytochemistry staining of undifferentiated hES cells (H9line) expressing OCT-3/4 marker protein expressed in their nuclei. Cellswere cultured with growth media on plasma polymerized plates coated withhuman fibronectin (FIG. 3B) and on TC plates coated with anextracellular matrix (such as, Matrigel™) (positive control, FIG. 3A).

FIG. 4 Quantitative Fluorescence Activated Cell Sorter (FACS) analysisof undifferentiated hES cell specific marker protein expression of cellsgrown on plasma polymerized plates coated with human fibronectin and onTC plates coated with an extracellular matrix (such as, Matrigel™)(positive control).

FIG. 5 Quantitative real time-polymerase chain reaction (QRT-PCR)analysis of germline-specific marker gene expression in embryoid bodiesgenerated from hES cells cultured on plama polymerized plates coatedwith human fibronectin and on TC plates coated with an extracellularmatrix (such as, Matrigel™) (positive control).

FIG. 6 Immunocytochemistry analysis of germline-specific marker proteinexpression in hES cell-derived differentiated cells. Cells wereimmunostained with DAPI and a germline-specific marker protein. Cellsexhibiting ectoderm-specific protein expression stained positively fornestin (FIG. 6A) or β-tubulin 3 (FIG. 6B). Cells exhibitingmesoderm-specific protein expression stained positively for α-sm actin(FIG. 6C) or brachyury (FIG. 6D).

FIG. 7 Graphical representation and pictoral of plates demonstratingconcentrating of hFN in the coating. FIG. 7A Comparison of boundfibronectin on plasma polymerized plates and TC plates coated withdifferent concentrations of human fibronectin and detected by anti-humanfibronectin ELISA. FIG. 7B Crystal violet staining of hES cells (H9line) grown for 3 days on human fibronectin-coated plasma polymerizedplates.

FIG. 8 Comparison of attachment and growth of human mesenchymal stemcells (MSC) on tissue culture plates in MSC media containing serum (FIG.8A); and on plasma polymerized plates coated with human fibronectin andcultured in serum free MSC media (FIG. 8B).

FIG. 9 Comparison of differentiation potential of human mesenchymal stemcells (MSCs) to adipocytes. MSCs at passage 5 were seeded on eitheruncoated tissue culture plates (FIG. 9A) or plasma polymerized platescoated with human fibronectin (FIG. 9B) and induced with adipogenicmedia. Cells plated on tissue culture plates were previously culturedwith serum containing media. Cells plated on plasma polymerized platescoated with fibronectin were previously cultured on the same surfacewith serum free MSC media.

FIG. 10 Comparison of hES cell derived neuronal stem cell attachment andgrowth on tissue culture and plasma polymerized plates. Cells wereseeded on uncoated tissue culture plates (FIG. 10 a), tissue cultureplates coated sequentially with polyornithine and laminin (FIG. 10 b),uncoated plasma polymerized plates (FIG. 10 c) and plasma polymerizedplates coated with human fibronectin (FIG. 10 d).

FIG. 11 Comparison of hES cell derived neuronal stem cell attachment andgrowth on tissue culture and plasma polymerized plates either uncoatedor coated with various ECM proteins.

FIG. 12 This graph depicts cell viability of hES cell derived neuronalstem cell using an MTS assay.

DETAILED DESCRIPTION OF THE INVENTION

A defined cell culturing substrate is provided for propagating stemcells in an undifferentiated state and maintaining their self-renewaland pluripotency characteristics for extended periods of time inculture. The defined culture surface of the present invention promotesmore efficient attachment and expansion of human embryonic as well asmesenchymal and neural stem cells in an undifferentiated state, ascompared to standard culture substrates such as tissue culture-treatedsurfaces. In some embodiments, hES cells, human bone marrow derivedmesenchymal stem cells and hESC-derived neuronal stem cells may bepropagated from the defined cell culture surface. In some embodiments,the defined cell culture surface is xeno-free.

A cell culture surface is provided. Preferably, the cell culture surfaceis defined on a culture vessel. The cell culture surface may be definedover media found within a cell culture vessel or other structure.Material for the cell culture surface may include plastic (e.g.polystyrene, acrylonitrile butadiene styrene, polycarbonate); glass,microporous filters (e.g., cellulose, nylon, glass fiber, polyester, andpolycarbonate); materials for bio-reactors used in batch or continuouscell culture or in genetic engineering (e.g., bioreactors), which mayinclude hollow fiber tubes or micro carrier beads;polytetrafluoroethylene (Teflon®), ceramics and related polymericmaterials. Any material listed above or others are suitable for use inthe present invention. The material for the cell culture surface may beis selected from: cellulose, polystyrene, polycarbonate,polytetrafluoroethylene, nylon, glass; polyethyleneterephthalate,polymethylpentane, polypropylene, polyethylene and combinations thereof.These materials may be porous or non-porous.

For illustrative purposes, reference shall be made herein to a cellculture or culture vessel. It is to be understood that the inventionherein may be utilized on various cell culture surfaces including, butnot limited to surfaces defined on media found in cell culture vessels,such as microbeads, microporous filters or other filtration or bindingmedia. Preferably, the cell culture surface is formed of polystyrene.

It is contemplated that any culture vessel that is useful for adherentcultures may be used. Preferred cell culture vessel configurationscontemplated by the present invention include multiwell plates (such as6-well, 12-well and 24-well plates), dishes (such as petri dishes), testtubes, culture flasks, roller bottles, tube or shaker flasks, and thelike.

The cell culture surface is coated with a plasma polymerized film. Thesource of the plasma polymerization is one or more monomers. Usefulpolymerizable monomers may include unsaturated organic compounds such asolefinic amines, halogenated olefins, olefinic carboxylic acids andcarboxylates, olefinic nitrile compounds, oxygenated olefins andolefinic hydrocarbons. In some embodiments, the olefins may includevinylic and allylic forms. In other embodiments, cyclic compounds suchas cyclohexane, cyclopentane and cyclopropane may be used.

As will be recognized by those skilled in the art, various plasmapolymerization techniques may be utilized to deposit the one or moremonomers onto the cell culture surfaces. Preferably, a positivelycharged polymerized film is deposited on the surfaces. As will beappreciated by one skilled in the art, the plasma polymerized surfacemay have a negative charge depending on the proteins to be usedtherewith. Amine is preferably used as the monomer source of thepolymer. In some embodiments, the plasma polymerized monomer is madeusing plasma sources to generate a gas discharge that provides energy toinitiate polymerization of gaseous monomers, and allows a thin polymerfilm to deposit on a culture vessel. Cyclic compounds may be utilizedwhich may include gas plasmas by glow discharge methods. Derivatives ofthese cyclic compounds, such as 1,2-diaminocyclohexane for instance, arealso commonly polymerizable in gas plasmas.

Particularly preferred are plasma polymerizable monomers includinghydroxyl, amine or carboxylic acid groups. The polymer film may beobtained from the group of carboxylic acid containing monomersconsisting of acrylic acid, methacrylic acid, acetic acid andvinylacetic acid including but not limited to vinyl-monomer containing acarboxylic acid that is polymerizable. Examples of typical aminemonomers include, fully saturated and unsaturated amine compounds up to20 carbon atoms (more typically 2 to 8 carbons). Ethylenicallyunsaturated compounds (especially primary, secondary or tertiary amines)include allylamine and saturated monomers include methylamine,propylamine, heptylamine and diaminopropane. Of these, particularlyadvantageous results have been obtained through use of allylamine anddiaminopropane.

Mixtures of polymerizable monomers may be used. Additionally,polymerizable monomers may be blended with other gases not generallyconsidered as polymerizable in themselves, examples being argon,nitrogen and hydrogen.

In one aspect of the invention, the polymer includes an amine co-polymer(polymerization of two or more monomers). The co-polymer is prepared bythe plasma polymerization of an organic amine with a saturated (alkane)or unsaturated (alkene, diene or alkyne) hydrocarbon. The hydrocarbonwould be of up to 20 carbons (but more usually of 4 to 8). Examples ofalkanes are butane, pentane and hexane. Examples of alkenes are buteneand pentene. An example of a diene is 1-7 octadiene. The co-monomer mayalso be aromatic-containing e.g. styrene.

Plasma polymerization may be carried out as a copolymer polymerizationof two components using any ratio of amine:hydrocarbon. Preferably, theamine:hydrocarbon ratio for the co-plasma polymerization is between thelimits of 100 (amine):0(hydrocarbon) to 20 (amine):80 (hydrocarbon) andany ratio between these limits.

With a plasma polymerized film coating deposited on the cell culturesurfaces, a coating composition is immobilized on the film-coatedsurface with a coating composition. The coating composition may includeone or more extracellular matrix (ECM) proteins and an aqueous solvent.The term “extracellular matrix” is recognized in the art. Its componentsinclude one or more of the following proteins: fibronectin, laminin,vitronectin, tenascin, entactin, thrombospondin, elastin, gelatin,collagen, fibrillin, merosin, anchorin, chondronectin, link protein,bone sialoprotein, osteocalcin, osteopontin, epinectin, hyaluronectin,undulin, epiligrin, and kalinin. Other extracellular matrix proteins aredescribed in Kleinman et al., J. Biometer. Sci. Polymer Edn., 5: 1-11,(1993), herein incorporated by reference. It is intended that the term“extracellular matrix” encompass a presently unknown extracellularmatrix that may be discovered in the future, since its characterizationas an extracellular matrix will be readily determinable by personsskilled in the art.

In some aspects, the total protein concentration in the coatingcomposition may be about 1 ng/mL to about 1 mg/mL. In some preferredembodiments, the total protein concentration in the coating compositionis about 1 g/mL to about 300 g/mL. In more preferred embodiments, thetotal protein concentration in the coating composition is about 5 g/mLto about 200 g/mL.

The extracellular matrix (ECM) proteins useful in the coating may be ofnatural origin and purified from human or animal tissues. Alternatively,the ECM proteins may be genetically engineered recombinant proteins orsynthetic in nature. The ECM proteins may be a whole protein or in theform of peptide fragments. Examples of ECM protein coatings that may beuseful in the coating include laminin, collagen I, collagen IV,fibronectin and vitronectin.

In some embodiments, the coating composition is xeno-free, in that theproteins are only of human origin. This may be desired for certainresearch applications.

In some embodiments, the coating composition includes syntheticallygenerated peptide fragments of fibronectin or recombinant fibronectin.

In still further embodiments, the coating composition includes a mixtureof at least fibronectin and vitronectin.

In some other embodiments, the coating composition preferably includeslaminin.

The aqueous solvent useful in preparing the coating compositions may bewater or a buffer, such as phosphate buffered saline, specificallyDulbecco's phosphate buffered saline (DPBS), or a cell culture media,for example. In some embodiments, DMEM, KO/DMEM, DMEM/F12, RPMI, orother cell culture media known in the art, are suitable for use as theaqueous solvent used to prepare the coating. Suitable aqueous solventdiluents can include any cell culture medium, which provides a conditionthat is compatible with embryonic cell culture, and preferably maintainsthe cells in a self-renewing and an undifferentiated state untildirected into a particular cell type in vitro. Such media may beobtained commercially, for example, from StemCell Technologies, Inc.(Vancouver, BC, Canada), Invitrogen Corporation (Carlsbad, Calif.) orSigma-Aldrich (St. Louis, Mo.).

The coating composition preferably includes a single type ofextracellular matrix protein. In some preferred embodiments, the coatingcomposition includes fibronectin, particularly for use with culturingstem cells. For example, a suitable coating composition may be preparedby diluting human fibronectin, such as human fibronectin sold by Becton,Dickinson & Co. of Franklin Lakes, N.J. (BD) (Cat#354008), in Dulbecco'sphosphate buffered saline (DPBS) to a protein concentration of 5 μg/mLto about 200 μg/mL.

In some other embodiments, the coating composition preferably includeslaminin. For example, a suitable coating composition may be prepared bydiluting laminin (Sigma-Aldrich (St. Louis, Mo.); Cat#L6274 and L2020)in Dulbecco's phosphate buffered saline (DPBS) to a proteinconcentration of 5 μg/ml to about 200 μg/ml.

In some embodiments, the coating composition has a pH of between about7.0 to about 8.5. The pH may be maintained with any buffering componentcapable of maintaining the composition within the pH range of about 7.0to 8.5. Potential buffer systems in this range include, but are notlimited to, diethanolamine, triethanolamine,(1,3-bis(tris[Hydroxymethyl]methylamino)propane);3-[N,N-bis(2-Hydroxyethyl)amino]-2-hydroxypropanesulfonic acid: DIPSO;(N-[2-Hydroxyethyl]piperazine-N′-[-4-butanesulfonic acid] HEPBS);(N-(4-(2-hydroxyethyl-1-piperazineethanesulfonic acid: HEPES);3-(N-Morpholino)butane sulfonic acid: MOBS);(piperazine-N,N′-bis[2-hydroxypropanesulfonic acid: POPSO);(N-tris(Hydroxymethyl)methyl-3-aminopropanesulfonic acid: TAPS;3-(N-tris[Hydroxymethyl]methylamino)-2-hydroxypropanesulfonic acid:TAPSO); (N-tris(Hydroxymethyl)methyl-2-aminoethanesulfonic acid: TES;(N-tris(Hydroxymethyl)methylglycine: Tricine; N-ethylmorpholine,dimethylleucylglycine, sodium 5:5-diethyl barbituate and 2 amino, 2methyl-1:3 propanediol.

The coating compositions used to prepare the culturing system of thepresent invention can include various components, which can affect theaccessibility of growth factors in the coating to cells and/or whichassist in cell adhesion and/or which affect the structure of theproteins in the coating. These components may include, but are notlimited to, salts, diluents, heparan sulfate proteoglycans.

A wide variety of other materials, may be included in the coating on thesubstrate. These include, but are not limited to, cells, antibodies,enzymes, receptors, growth factors, additional components of theextracellular matrix, cytokines, hormones and drugs. In someembodiments, the extracellular matrix proteins can bind to thesematerials. These biologically active materials, if present, can bereadily available to the cultured cells to moderate or regulate theirproperties or behavior.

The present invention provides a method of preparing a stable,ready-to-use cell-culturing system. This method includes applying anextracellular matrix coating composition to a cell culture surfacecoated with plasma polymerized film, wherein the total proteinconcentration in the coating composition is about 1 ng/mL to about 1mg/mL. The method also includes immobilizing proteins in the coatingcomposition on such film-coated vessel over a period of time; andremoving the excess coating composition from the film-coated plates.

The coating composition is generally applied in the followingquantities: approximately 0.5 to 2.0 mL of the coating composition maybe applied to a well in a 6-well multiwell plate; about 0.25 to 1.0 mLmay be applied to a well in a 12-well or 24-well multiwell plate; about50 μL to 100 μL may be applied to a well in a 96-well plate; about 0.5to 2.0 mL may be applied to a 35 mm dish; about 0.5 to 4.0 mL may beapplied to a 60 mm dish; about 2.0 to 12.0 mL may be applied to a 100 mmdish; about 0.5 mL to 4.0 mL may be added to a T25 flask (having 25 cm²cell attachment surface); about 2.0 mL to 12.0 mL may be added to a T75flask (having 75 cm² cell attachment surface); and, about 5.0 mL to 25.0mL may be added to a T175 flask (having 175 cm² cell attachmentsurface).

After application, the coating composition is maintained on thefilm-coated surface to permit adsorption of the extracellular matrixproteins in the composition to the plasma polymerized plates. Thecoating composition may be maintained in an uncontrolled environment(e.g., room temperature) or a controlled environment (e.g., heated orchilled conditions). In particular, coated plasma polymerized plates aredesirably incubated at temperatures from about 22° C. to about 37° C.,and for a period of time of about 30 minutes to about 4 hours to permitadsorption of the proteins to the substrate surface. Alternatively, thecoated plasma polymerized plates may be incubated at 4° C. overnight to2 weeks prior to use. The excess coating composition is removed from thecoated substrate immediately prior to use for cell culture to remove theunadsorbed proteins and remaining solution.

The cell culturing systems of the present invention can be used invarious applications, including culturing of embryonic stem (ES) cells,mesenchymal and neuronal stem cells. In a preferred embodiment, axeno-free, defined cell culture substrate may be useful for maintainingthe self-renewal and pluripotency characteristics of undifferentiated ESand adult stem cells for extended periods of time.

In some embodiments, the ES cells, particularly human ES (hES) cells,may be cultured using a cell culturing vessel of the subject invention.For example, hES cells include, but are not limited to, the followingcell lines: H1, H9, and H14, for example. These cell lines areavailable, for example, from WiCell Research Institute, Madison, Wis.

The cell culturing surface of the subject invention may be used toculture stem cells. The method may include culturing embryonic stemcells and providing a cell culturing system including a culture vesselwith a plasma polymerized surface; and a coating thereon of a coatingcomposition. The coating composition includes a mixture of extracellularmatrix proteins and an aqueous solvent, the total protein concentrationin the coating composition being about 1 ng/mL to about 1 mg/mL. Theculturing method may also include adding a suspension of embryonic stemcells to the cell culturing system; and incubating the embryonic stemcells at 5% CO₂ in humidified air at 37° C. to produce undifferentiatedcolonies for embryonic stem cell expansion.

In further embodiments, a culture medium is utilized in the cellcultures. The culture medium may include base media and supplements toassist in the adherence of stem cells to the cell culturing substrate.In some embodiments, a culture medium such as mTeSR™ 1 (StemCellTechnologies Inc.) may be included. It is noted, however, that themethod of culturing is not limited to this culture medium.

In some embodiments, adult stem cells, which may be mesenchymal stemcells, may be cultured with the subject invention. For example,mesenchymal stem cells may include, but are not limited to, bone marrowderived cells such as Poietics® Human Mesenchymal Stem Cells. Thesecells are available, for example, from Lonza (Wakersville, Md.).

In further embodiments, the culture medium used to culture themesenchymal stem cells is a serum free culture media such as STEMPRO®MSC SFM (Invitrogen Corporation, Carlsbad, Calif.). It is noted,however, that the method of culturing is not limited to this culturemedium.

In other embodiments, neuronal stem cells, which may be hES cellderived, may be cultured with the subject invention.

In further embodiments, the culture medium used to culture the hES cellderived neuronal stem cells is a serum free culture media onDMEM/F12+Glutamax, N2, B27, bFGF and Pen/Strep.

The culture system of the present invention can be used to test variousinhibitors or stimulators to determine their effectiveness in a cellstudy. Stimulators can include growth factors, which are known in theart. For example, these can include one or more of platelet derivedgrowth factors (PDGF), e.g., PDGF AA, PDGF BB; insulin-like growthfactors (IGF), e.g., IGF-I, IGF-II; fibroblast growth factors (FGF),e.g., acidic FGF, basic FGF, β-endothelial cell growth factor, FGF 4,FGF 5, FGF 6, FGF 7, FGF 8, and FGF 9; transforming growth factors(TGF), e.g., TGF-P1, TGF β1.2, TGF-β2, TGF-β 3, TGF-β 5; bonemorphogenic proteins (BMP), e.g., BMP 1, BMP 2, BMP 3, BMP 4; vascularendothelial growth factors (VEGF), e.g., VEGF, placenta growth factor;epidermal growth factors (EGF), e.g., EGF, amphiregulin, betacellulin,heparin binding EGF; interleukins, e.g., IL-1, IL-2, IL-3, IL-4, IL-5,IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14; colonystimulating factors (CSF), e.g., CSF-G, CSF-GM, CSF-M; nerve growthfactor (NGF); stem cell factor; hepatocyte growth factor, and ciliaryneurotrophic factor. Additional growth factors are described in Spornand Roberts, Peptide Growth Factors and Their Receptors I,Springer-Verlag, New York (1990), which is hereby incorporated byreference. The term “growth factors” is intended to encompass presentlyunknown growth factors that may be discovered in the future, since theircharacterization as a growth factor will be readily determinable bypersons skilled in the art.

In the embodiments of the present invention, the culture medium may besupplemented with serum, but is preferably serum-free. The culturemedium may be a medium that is previously conditioned by exposure tofibroblast feeder layer cells (i.e. feeder conditioned medium). Asuitable, defined serum-free medium, mTeSR™1, for culturing humanembryonic stem cells is available from StemCell Technologies, Inc. Asuitable, serum-free medium for culturing bone marrow derivedmesenchymal stem cells, STEMPRO® MSC SFM, is available from InvitrogenCorporation (Carlsbad, Calif.).

Methods of Preparation

The present invention provides a method of preparing a stable,ready-to-use cell culturing substrate. This method includes applying anextracellular matrix coating solution to a plasma polymerizedfilm-coated surface of a cell culture vessel, wherein the total proteinconcentration in the coating solution is about 1 ng/ml to about 1 mg/ml.The method also includes maintaining the coating solution on thefilm-coated surface so as to allow immobilizing of proteins from thecoating solution on the substrate surface; and removing the excesscoating solution from the substrate. In some aspects, one or morecoating solutions may be added to the plasma polymerized film-coatedsurface.

In some embodiments coating solutions, with the same or different ECMproteins, may be serially applied. An optional washing step may beutilized between coating applications. In some embodiments, the washingstep(s) may include washing the substrate with distilled water, a buffer(e.g., PBS) or a culture medium. In other embodiments, the washingstep(s) may include washing the substrate with a blocking solution.

The coating solution, including the extracellular matrix components, canbe maintained on at least one surface of a film-coated cell culturingsurface, such as a the surface of a cell culturing vessel (e.g., flaskor cell culture plate), at a temperature and/or time period sufficientto allow adsorption of the extracellular matrix proteins to the coatedsurface. For example, the film-coated cell culturing vessel can bemaintained at room temperature from about 1 to about 4 hours to allowadsorption. Alternatively, the container can be incubated at 4° C. overa period of time (e.g. 4 hours to 2 weeks). Also, coated substrates maybe incubated at temperatures from about 22° C. to about 37° C., and fora period of time of about 30 minutes to about 4 hours to permitadsorption of the proteins to the substrate surface. Any excess coatingsolution is thereafter removed (e.g., by aspiration), and the coatedsubstrate may be washed with an aqueous solvent (e.g., water, buffer,ddH₂O, culture media) to remove unbound proteins. Use of a blockingsolution, as described above, increases the stability of the coatedsubstrate.

After the coating is applied, it can optionally be sterilized. In oneembodiment, the apparatus is sterilized using ultra-violet (UV) light.

In some aspects, the cell culturing substrate may be frozen immediatelyafter the coating solution is applied to the film-coated surface. Thefrozen cell culturing substrate may be stored at −20° C. for up to 3months and then thawed prior to use. For example, laminin as the ECMprotein may be applied as a coating solution, as described above, withthe resulting cell culturing surface being frozen and stored.

The following examples are for illustrative purposes and are notintended to, in any way to limit the embodiments and uses of the presentinvention.

EXAMPLES Example 1 ECM Coating on Chemically Defined Plasma PolymerizedSurfaces

Chemically defined plasma polymerized culture vessels (12 or 6 multiwellplate format) were coated with various extracellular matrix (ECM)proteins. Individual or combined ECM proteins, diluted in Dulbecco'sphosphate buffered saline (DPBS) or DMEM/F12 media, were added (1mL/well for 6 well plates and 0.5 mL/well for 12 or 24 well plates) andthe plates were coated for 2 hours at room temperature or at 37° C.Coating solution was removed immediately prior to use for hES cellculture experiments. The following ECMs were used in the experiment:ECMs include but are not limited to human fibronectin, human laminin,human vitronectin, human collagen IV, BD Matrigel™ hESC-qualifiedMatrix, ProNectin® F Plus; a fibronectin-like engineered protein polymerfrom Sigma; Retronectin another recombinant fragment of humanfibronectin from Takara Bio USA.

Plasma polymerized 6 well plates BD Primex 1, (which includes amines anda positively charged surface, Cat #359296) and BD Primex 2 (whichincludes carboxyls and a negatively charged surface, Cat #359297), thatare chemically defined, were coated with various animal free ECMs.Plasma polymerized 24 well plates (BD PureCoat amine, a positivelycharged plasma polymerized surface, Cat #354723 or 356723), that arechemically defined, were coated with various human and animal derivedECMs.

The coating solution was made by diluting single or a mixture of ECMproteins in Dulbecco's phosphate buffered saline at a final proteinconcentration of 5 μg/mL to 50 μg/mL.

The coating solution was added at a volume of 1 mL per well for a 6 wellplate or 0.5 mL per well for a 12 well plate.

The coating solution was incubated on plasma polymerized plates for aminimum of 2 hours at room temperature or overnight at 4° C.

The coating solution was removed immediately prior to use of the platesfor hES cell culturing experiments.

Example 2 Culture of Human Embryonic Stem Cells on ECM Coated PlasmaPolymerized Surfaces hES Cell Culture on ECM-Coated Plasma PolymerizedSurface

hES cells (H1, H9 or H14 lines from WiCell Institute) were initiallyplated onto plasma polymerized plates (with or without ECM coating) frompositive control plates (i.e. hESC cells grown on 6 well TC platescoated with BD Matrigel™ hESC-qualified Matrix and grown in mTeSR™ 1medium).

Cells on positive control plates were treated with dispase (2 mg/mL) for5 minutes at 37° C., followed by four quick washes with DMEM/F12 mediumand then mechanically dissected to small clumps in a small volume ofmTeSR™1 medium using plastic pipettes or pipette tips. Clumps of hEScells resuspended in mTeSR™1 medium were seeded onto test surfaces at a1:3 to 1:6 split ratio and cultured in an incubator at 5% CO₂ inhumidified air at 37° C. Culture medium was replaced daily and cellswere typically dissociated every 4 to 6 days after initial plating.

hESC Dissociation on Plasma Polymerized BD Primex 1 Plates

hES cells cultured on BD Primex 1 plates coated with BD humanfibronectin were routine dissociated with TryPLE select (Invitrogen,diluted 1:2 (v:v) with DMEM/F12) for sub-culturing and long-termmaintenance. Briefly, spent culture medium was removed, cells wererinsed once with DMEM/F12 medium, and treated with diluted TryPLE select(1 mL/well) for 2 minutes at room temperature. Dissociation reagent wasthen promptly removed and cells were washed three times in rapidsuccession with DMEM/F12 medium. mTeSR™1 medium (StemCell Technologies,Inc.) was then added to the treated cells and colonies were mechanicallydissected to small clumps using a 5 mL plastic pipette or pipette tips.Dissociation and plating of cells were then carried out as described inthe above section.

hES Cell Colony Attachment Assay

Attachment of hES cells on plasma polymerized surfaces coated withvarious ECM proteins were compared.

hES cells were fixed for 20 minutes with 4% paraformaldehyde at roomtemperature followed by two washes with Dulbecco's phosphate bufferedsaline (DBPS) for 5 minutes each. Cells were then stained for 5 minuteswith crystal violet stain (diluted 1:10 with DPBS) and washed once withDPBS. The plates containing fixed and crystal violet stained cells werescanned using a laser scanner and the attached colonies per well werevisualized (FIG. 1). FIG. 1A is (TC)-treated polystyrene plates coatedwith BD Matrigel™ hESC-qualified matrix, FIG. 1B is TC plates coatedwith BD human fibronectin; and FIG. 1C BD Primex 1 plates (Cat #359296)coated with BD human fibronectin and cultured with mTeSR™1 medium(StemCell Technologies, Inc.).

Colony attachment and morphology of the cells were also routinelymonitored using a phase contrast microscope. As evidenced by FIG. 2A andFIG. 2B, hES cells (H9 line) were grown on plasma polymerized BD Primex1 plates (Cat #359296) coated with BD human fibronectin for 18 passages(FIG. 2B). Cell morphology on the BD Primex 1 plates coated withfibronectin was very similar to hES cell colonies grown on positivecontrol substrate, TC plates coated with BD Matrigel™ hESC-qualifiedmatrix (FIG. 2A). hES cells maintained a predominantly undifferentiatedstate when cultured on both of these substrates. Results

Human embryonic stem (hES) cells (H9 line) were seeded on tissue culture(TC)-treated polystyrene plates coated with BD Matrigel™ hESC-qualifiedmatrix or human fibronectin; and on BD Primex 1 coated with humanfibronectin. Cells were and cultured with mTeSR™1 medium (StemCellTechnologies, Inc) for four days, fixed and stained with crystal violet.Relative cell attachment on each surface is shown in FIG. 1. As can beseen colony attachment on BD Primex 1 coated with human fibronectin(FIG. 1C) is comparable to positive control substrate (TC plates coatedwith BD Matrigel™ hESC-qualified matrix, FIG. 1A). However, TC surfacecoated with human fibronectin did not support appreciable hES cellcolony attachment or growth with mTeSR™1 medium (FIG. 1B).

Results of hES cell colony attachment is shown below on BD Primex 1 andTC plates with or without various types of human fibronectin proteincoating:

TABLE 1 BD Primex 1 TC Uncoated 0 0 BD Matrigel ™ 3 3 hESC-qualifiedMatrix BD fibronectin 3 1 (from human plasma) Sigma fibronectin 3 Nottested (from human plasma) Sigma fibronectin 2 Not tested (from humanForeskin Fibroblast) Retronectin 3 Not tested (recombinant Fragment ofhuman fibronectin from Takara Bio USA) Pronectin ® F Plus 1 Not tested(human fibronectin-like engineered protein polymer, Sigma) COLONYATTACHMENT SCALE 0: no colony attached 1: low (~1 to 10 colonies) 2:moderate (~10-20 colonies) 3: high (typically >20 colonies andcomparable to positive control Positive control = TC surface coated withpre-qualified BD Matrigel ™

Surface was considered comparable to positive control based onapproximate number of colony attachment, cellular morphology within thecolonies (i.e. compact vs. single cells), rate of proliferation anddegree of spontaneous differentiation.

Results of hES cell colony attachment is shown below on plasmapolymerized BD Primex 1 and BD Primex 2 plates with or without variousECM protein coating:

TABLE 2 Plasma polymerized surface BD Primex 2 BD Primex 1 Uncoatedsurface 0 0 Sigma human Laminin 3 2 BD human fibronectin 2 3 BD humanfibronectin + Not 2 BD human collagen IV tested BD human fibronectin +Not 3 human vitronectin tested BD Human Matrix 1 1 COLONY ATTACHMENTSCALE 0: No colony attached 1: low (~1 to 10 colonies) 2: moderate(~10-20 colonies) 3: high (typically >20 colonies)

Surface was considered comparable to positive control based onapproximate number of colony attachment, cellular morphology within thecolonies (i.e. compact vs. single cells), rate of proliferation anddegree of spontaneous differentiation.

Example 3 Characterization of Undifferentiated hESCs

Morphological analysis (FIG. 2) reveals that hES cells maintained apredominantly undifferentiated state when cultured with mTeSR™1 on BDPrimex 1 plates (Cat #359296) coated with BD human Fibronectin (FIG. 2B)and was comparable to those cultured on positive control substrate (TCplates coated with BD Matrigel™ hESC-qualified matrix, FIG. 2A).

Expression of the undifferentiated marker OCT-3/4 was comparable forcells cultured with mTeSR™1 on BD Primex 1 plates coated with BD humanfibronectin (FIG. 3B) and on positive control substrate (TC platescoated with BD Matrigel™ hESC-qualified matrix, FIG. 3A).

Quantitative FACS analysis (protocol outlined below in Example 5)revealed that expression of undifferentiated hES cell-specific markerexpression (OCT-3/4 and SSEA-4) for hES cells (H9 line) cultured on BDPrimex 1 coated with BD human fibronectin for sixteen passages werecomparable to positive control cells (cultured on TC coated with BDMatrigel™ hESC-qualified matrix). The relative percentage of cells thatexpressed undifferentiated markers are summarized in Table 3 below.

TABLE 3 Cells positive for undifferentiated markers (%) Surface OCT-4SSEA-4 BD Primex 1 86.82 95.58 TC + BD Matrigel ™ 87.2 90.95

Example 4 Inducing Spontaneous Differentiation by Embryoid BodyFormation

hESC colonies were dissociated with either TryPLE select (Invitrogen) inthe same manner as described above in Example 2.

Dissociated cell clumps were plated on petri dishes (not tissue culturetreated) or low attachment 6 well plates in differentiation medium(DMEM/F12 medium supplemented with 20% fetal bovine serum (FBS), 10 mMnon-essential amino acids, 1 mM L-glutamine, 0.1 mMbeta-mercaptoethanol).

Cell clumps cultured in differentiation medium formed embryoid bodies(EBs) in suspension and were grown for 4-15 days. Subsequently the EBswere either analyzed by QRT-PCR (protocol known in prior art) for germlayer marker gene expression; or re-plated on gelatin-coated TC plates,further differentiated in DMEM supplemented with 20% FBS for additionalperiods of time, and analyzed using immunohistochemistry for presence ofgermlayer specific protein expression.

Example 5 Characterization of Pluripotency of hESCs

H9 hES cells (following culture for 13 passages on BD Primex 1 platescoated with BD human fibronectin) were differentiated into embryoidbodies (EBs). Expression of 3 germ layer markers in EBs was determinedby QRT-PCR (FIG. 5). All three germ layer markers FoxA2 (forkhead box A2expressed in Endoderm), HAND1 (heart and neural crest derivativesexpressed 1 in Mesoderm) and tubulin TUBB3 (tubulin beta 3 in Ectoderm)were detected. Cells grown on TC plates coated with BD Matrigel™hESC-qualified Matrix represents positive control. Expression of thegerm layer markers relative to undifferentiated cells cultured on BDPrimex 1 plates coated with BD human fibronectin (control) is alsoshown. As can be seen, there is a significant increase in expression ofall 3 germ layer markers for EBs generated from cells cultured on BDPrimex 1 plates coated with BD human fibronectin and expression iscomparable to positive control. The above data suggest that the cellscultured on Primex 1 with fibronectin coating maintain theirpluripotency.

Germ layer marker protein expression after spontaneous differentiationfollowing EB formation is shown in FIG. 6. hES cells (H9 line) werecultured on BD Primex 1 plates coated with BD human fibronectin for 3passages. Cells were differentiated into EBs for 10 days and transferredto gelatin-coated TC plates and cultured for 10 additional days in DMEMmedium supplemented with 20% FBS. Expression of germ layer markers wasdetermined by immunohistochemistry.

Example 6 Immunocytochemistry Protocols

The present example is directed to an immunocytochemistry protocol usedto test for marker expression of undifferentiated hES cells (dataoutlined in Example 3 and shown in FIG. 3). The present protocol is forcells grown on 6-well plates. The same protocol was also used fordetecting expression of germ layer specific proteins in differentiatedhES cells (data outlined in example 4 and shown in FIG. 6). For thelatter study, cells were grown on 12-well plates and half of the volumesindicated in the following protocol were used per well.

Cultured hES cells were washed with 2 mL of Dulbecco's phosphatebuffered saline (DPBS). Then, the cells were fixed with 1 mL of 4%paraformaldehyde for 20 minutes at room temperature. The fixed cellswere washed twice with 2 mL of DPBS for 5 minutes each. Subsequently,the cells were blocked with 1 mL of 0.1% bovine serum albumin (BSA) and10% normal goat serum in DPBS. During the blocking step, the primaryantibody working solution was prepared with DPBS containing 1% BSA and10% normal goat serum to a final desired antibody concentration. It isnoted, that for both the blocking solution, and the primary antibodysolution, the normal serum may be replaced with that from anotherspecies depending on the host species of the secondary antibody.

After blocking, the cells were incubated with 1 mL/well of the dilutedantibody working solution for 2 hours at room temperature or overnightat 2-8° C. Then, the cells were washed three times with 2 mL of DPBScontaining 1% BSA for 5 minutes each wash.

The secondary antibody was diluted 1:2000 in DPBS containing 1% BSA.Useful fluorescent secondary antibodies included Alex 488 or594-conjugated appropriate secondary antibodies (Invitrogen-MolecularProbes). The cells were incubated with the diluted secondary antibody 1mL/well for 60 minutes at room temperature in the dark. Subsequently,the cells were washed three times with 2 mL of DPBS containing 1% BSAfor 5 minutes each wash. The cells were thereafter covered with 2 mL ofDPBS and visualized and imaged with a fluorescent microscope.

Example 7 FACS Analysis Protocol

Confluent cultures of human embryonic stem (hES) cells (H9 line) grownon 6 well plates were briefly rinsed with Dulbecco's phosphate bufferedsaline (DPBS) and treated with 1 mL/well 0.25% Trypsin/EDTA (Invitrogen)for 2-3 minutes at 37° C. to dissociate the cells from the surface.Trypsin was inactivated by adding 2 mls/well DMEM/F12 medium containing20% fetal bovine serum (FBS). Cells were then gently triturated bypipetting up and down, and pelleted by centrifugation at 1000 rpm for 5minutes.

For intracellular antigens (e.g. OCT-3/4), the cell pellet wasresuspended and fixed in 1% paraformaldehyde (1 mL/tube) for 10 mins at37° C. Fixed cells were washed one briefly with 3 mL/well perm/washbuffer (BD Pharmingen). Cells were pelleted by centrifugation, thesupernant was discarded and the cell pellet was resuspended in 3 mL/wellperm/wash buffer and incubated on ice for 15 minutes to permeabilize thecells. Following permeabilization, cells were pelleted once more bycentrifugation and resuspended in 100 μl of perm/wash buffer and probedwith appropriate primary antibodies (˜3×10⁵ cells incubated with 500 ngof primary antibody). Following 1 hour of incubation, cells were washedwith 3 mL of perm/wash buffer and resuspended in 100 μL of the samebuffer. Secondary antibody was added and cells were incubated for 30minutes in the dark at room temperature. Following incubations, cellswere washed as above and resuspended in 200 μL of perm/wash buffercontaining 1 μg/ml of propidium iodide (Sigma) to identify viable cells.Fluorescence-activated cell sorter (FACS) analysis was performed byusing a FACS Calibur Flow Cytometer (BD). A total of 30,000 events wereanalyzed per sample and data was analyzed using CellQuest 3.0 software(BD).

For surface antigens (such as SSEA-4, TRA-1-60, TRA-1-81 etc.) a similarprotocol was followed with some exceptions. Following dissociation,cells were resuspended in 0.5 mL of DPBS containing 25% FBS. Thefixation step was omitted for detection of surface antigens. All primaryantibody incubations were carried out in DPBS containing 25% FBS on iceusing 200 ng antibody per reaction, while secondary antibody incubationwas carried out in the same buffer for 30 minutes in the dark at roomtemperature. All other steps were similar to the protocol outlined forintracellular antigens above.

Example 8 Fibronectin ELISA Assay

The performance of the ECM-coated BD Primex plates was further definedby the results of an ELISA assay for human fibronectin. For example, afibronectin ELISA or laminin ELISA may be used to assess the amount ofthe ECM protein adsorbed on the substrate surface. By way of example, ahuman fibronectin ELISA assay, which was used to assess performance ofthe coated vessels is set forth below. A graph of fibronectin ELISA datafor BD Primex 1 plates and TC plates coated with varying concentrationsof human fibronectin is shown in FIG. 7.

In order to prepare a working primary antibody solution for the ELISA,0.5 ml of a 1:100 (v/v) stock solution of rabbit anti-human fibronectin(Sigma; catalog no. F3648) was added to 40 ml of 0.5% bovine serumalbumin (BSA) in Dulbecco's phosphate buffered saline (DPBS). In orderto prepare a working solution of secondary antibody for the ELISA, 0.4ml of a 1:100 (v/v) stock solution of goat anti-rabbit IgG-HRP (BDPharmingen; catalog no. 554021) was added to 40 ml of 0.5% BSA in DPBS.The ELISA was performed using: ECM-coated 6-well BD Primex 1 platesprepared according to the present invention (test plates), TC platescoated with BD Matrigel™ hESC-qualified Matrix (positive control) orFalcon 6-well uncoated plates (catalog no. 353046; negative control).The plates were first washed 3 times with 2 mL/well wash buffer (DPBSwith 0.02% Tween-20). Then, 1 mL of 0.5% BSA in DPBS was added as ablocking solution, and the plates were incubated at room temperature for1 hour. The BSA solution was then removed, and the plates were washedwith wash buffer as described above. Next, 1 mL/well of the primaryantibody working solution was added, and the plates were incubated for 2hours at room temperature. After removing the primary antibody solution,the plates were again washed as described above. Subsequently, 1 mL/wellof the secondary antibody working solution was added, and the plateswere incubated at room temperature for 1 hour. After removing thesecondary antibody solution, the plates were again washed as describedabove. Next, 1 ml/well of horseradish peroxidase (HRP) substrate, TMB(KPL; catalog no. 53-00-02) was added, and blue color was allowed todevelop for 8 minutes. Subsequently, 1 mL of stop solution (KPL; catalogno. 50-85-05) was added, and the plates were swirled gently in order tofacilitate mixing. Then, a 200 μl aliquot from each well of the 6-wellplate was transferred to wells of a 96-well plate, and the absorbancewas measured at 450 nm at room temperature using a spectrophotometer(SpectraMax® Plus384, Molecular Devices). The plates were read within 5minutes after the stop solution was added. The mean absorbance per wellwas calculated for the test plates and positive control TC plates.

Results of Fibronectin ELISA Assay:

No significant difference in the amount of fibronectin attachment wasdetected between plasma polymerized BD Primex 1 plates as compared to TCplates (FIG. 7A). However, hES cell attachment and growth is supportedon BD Primex 1 coated with BD human fibronectin (FIG. 1C and 7B), and isnot optimal on TC surface coated with BD human fibronectin (FIG. 1B).Hence there is no obvious correlation between concentration offibronectin as detected by ELISA data and hES cell attachment and growthon BD Primex 1 plates.

Human fibronectin concentration of 5 to 50 μg/mL supports goodattachment and growth of hES cells on Primex 1 (FIG. 7B). However, closeinspection of colony morphology suggests that 10-50 μg/mL is the bestrange for long-term culture of these cells.

Collectively this data suggest that it is not the amount of boundfibronectin, but rather the conformation of this ECM protein, thatoffers an advantage on plasma polymerized BD Primex 1 plates for hEScell attachment and growth.

Example 9 Mesenchymal Stem Cell (MSC) Attachment and Growth in SerumFree Media on ECM Coated Plasma Polymerized Surfaces

Bone marrow derived MSCs (Lonza) were thawed and expanded on tissueculture flasks with complete MSC growth medium containing 10% serum(MSCGM™, Lonza). At passage 5, cells were dissociated with 0.5% trypsinEDTA, and plated on uncoated tissue culture plates with MSC growthmedium containing serum (Lonza) (positive control; FIG. 9A) or on BDprimex 1 plates coated with fibronectin in serum-free STEMPRO® MSCmedium (Invitrogen) (FIG. 9B). After 5 days in culture, cell morphologyand growth were visually inspected using a microscope. Typicallyattachment and growth of MSCs in serum-free medium is poor. In thisexample, it has been demonstrated that attachment of MSCs on BD Primex 1plates coated with fibronectin is comparable to positive controlconditions (cells grown on tissue culture surface with media containingserum).

Example 10 MSC Differentiation to Adipocytes on ECM Coated PlasmaPolymerized Surfaces

Adipogenesis Culture Protocol

Adipogenic Induction Medium and Adipogenic Maintenance Medium werepurchased from Lonza and manufacturer's protocol for adipogenesis wasfollowed.

200,000 mesenchymal cells per well in 2 mL of medium were plated in 6well tissue culture plates with serum containing growth media (MSCGM™,Lonza) or on fibronectin coated BD Primex 1 plates with serum-free MSCmedia (STEMPRO® MSC SFM; Invitrogen). Cells were Incubated at 37° C., ina humidified atmosphere of 5% CO2. Media was replaced on cells every 2to 3 days until the cultures reached confluence (in ˜7 days). At 100%confluence, three cycles of induction/maintenance were followed tostimulate adipogenic differentiation. Each cycle consisted of feedingthe MSCs with Adipogenesis Induction Medium and cultured for 3 days (37°C., 5% CO2) followed by 1 to 3 days of culture in Adipogenic MaintenanceMedium. After 3 complete cycles of induction/maintenance, the MSCs werecultured for 7 more days in Adipogenic Maintenance Medium, and mediumwas replaced every 2-3 days. The extent of adipogenic differentiationwas visually inspected using a microscope to determine the presence oflipid vacuoles in the induced cells.

Results of MSC Differentiation to Adipocytes

The extent of adipogenesis on BD primex 1 plates coated with fibronectin(FIG. 9B) was similar to that observed on positive control tissueculture plates (FIG. 9A). This example illustrates that MSCs culturedwith serum-free media on BD primex 1 plates coated with fibronectinretain their ability to differentiate into adipocytes anddifferentiation potential is comparable to that observed with positivecontrol.

Example 11 NSC Growth and Attachment on ECM Coated Plasma PolymerizedSurfaces

Human embryonic stem cell derived neuronal stem cells (hNSCs) werecultured in DMEM/F12 Media (1:1) supplemented with 2.5 mM L-Glutamine,1% N2, 2% B27, 20 ng/mL bFGF and 1% Pen/Strep on tissue culture flaskscoated sequentially with polyornithine followed by laminin.

To coat T-75 tissue culture flasks, 5 mL of polyornithine (20 μg/mL)dissolved in distilled water was added and flasks were laid flat toensure the coating solutions evenly covered the bottom surface. Flaskswere incubated overnight at room temperature. After 24 hours, thepolyornithine solution was removed, the flask was rinsed once withdistilled water and 5 mL of laminin (5 μg/mL) dissolved in Dulbecco'sphosphate buffered saline was added. The bottom surface of the flask wascoated with laminin and incubated at 37° C. for 2 hours. The coatingsolution was removed immediately prior to use for plating hNSCs.

In the present example, attachment and growth of hNSCs on ECM coatedplasma polymerized surface was tested. Six well tissue culture and BDPrimex 1 plates were coated with BD human fibronectin (25 μg/mL) for 2hours at room temperature or a combination of polyornithine (20 μg/mL)and laminin (5 μg/mL) as described above. Tissue culture and BD PureCoatAmine plates (24 well) were coated with either polyornithine (20 μg/mL),laminin (5 μg/mL) or a combination of polyornithine (20 μg/mL) andlaminin (5 μg/mL) as described above.

FIG. 10 illustrates that attachment and growth of hNSCs on BD primex 1coated with fibronectin (FIG. 10 d) is equivalent to tissue culturesurface coated with a combination of polyornithine and laminin (positivecontrol, FIG. 10 b). Whereas, attachment and growth of hNSCs on uncoatedtissue culture surface or BD Primex 1 is very low (FIGS. 10 a and 10 crespectively).

FIG. 11 illustrates that attachment and growth of hNSCs on BD PureCoatAmine coated with laminin (5 μg/mL) or a combination of polyornithineand laminin was better than tissue culture surface coated with the sameECMs.

Example 12 NSC Viability was Determined Using an MTS Assay

A tetrazolium-based assay was utilized to quantify cell viability.Briefly, exhausted media was removed, replaced with media containing MTSreagent (Promega), and incubated at 37° C. for 2 hours. MTS is atetrazolium compound that is reduced by metabolically active livingcells into a soluble product, formazan, that gives a purple hue.

The absorbance of formazan at 490 nm was then read on a Tecan® Safire2™microplate reader.

hNSCs were seeded at a density of 20,000 cells/well in 24 well BDPureCoat Amine plates where wells were either uncoated or coated withpolyornithine, laminin or a combination of polyornithine and laminin.Three days later, spent medium was gently aspirated and replaced with400 μL of fresh growth medium without bFGF. To each well, 80 μL of MTSreagent added and cells were incubated for 1.5 hours at 37° C.Absorbance was read at 490 nm

Results of this experiment support the visual observations described inExample 8 (FIG. 12). hNSC viability on the BD PureCoat Amine surface wasapproximately 20 to 30% higher compared to tissue culture surface whencoated with either laminin alone or with a combination of polyornithineand laminin. This example demonstrates that it is possible for hNSCs toattach, grow and remain viable on BD PureCoat Amine surface with asingle ECM (laminin) and that it outperforms growth of these cells ontissue culture plates with two ECMs (polyornithine and iaminin; positivecontrol).

What is claimed is:
 1. A cell culturing substrate comprising: a cellculture surface having a film attached thereto, wherein said filmcomprises one or more plasma polymerized monomers; and a coating on saidfilm-coated surface, said coating deposited from a coating solutioncomprising one or more extracellular matrix proteins and an aqueoussolvent, wherein the total extracellular matrix protein concentration inthe coating solution is about 1 ng/mL to about 1 mg/mL.
 2. The cellculturing substrate of claim 1, wherein said one or more monomers isselected from the group consisting of acrylic acid, methacrylic acid,acetic acid, vinylacetic acid and combinations thereof.
 3. The cellculturing substrate of claim 1, wherein said one or more monomers isselected from the group consisting of allylamine, methylamine,propylamine, heptylamine and diaminopropane.
 4. The cell culturingsubstrate of claim 1, wherein said monomer is selected from the groupconsisting of alkanes, alkenes, dienes, styrenes and combinationsthereof.
 5. The cell culturing substrate of claim 1, wherein said one ormore monomers is selected from the group consisting of amines,hydrocarbons and combinations thereof, wherein the ratio of amine tohydrocarbon is between about 100:0 and about 20:80.
 6. The cellculturing substrate of claim 1, wherein the total protein concentrationin the coating composition is about 1 μg/mL to about 300 μg/mL.
 7. Thecell culturing substrate of claim 1, wherein the total proteinconcentration in the coating composition is about 5 μg/mL to about 200μg/mL.
 8. The cell culturing substrate of claim 1, wherein the coatingcomposition comprises extracellular matrix proteins selected from thegroup consisting of natural, recombinant, synthetic extracellular matrixproteins and combinations thereof.
 9. The cell culturing substrate ofclaim 1, wherein the coating composition comprises a whole extracellular matrix protein or a fragment of the extracellular matrixprotein
 10. The cell culturing substrate of claim 1, wherein the coatingcomposition further comprises a component selected from the groupconsisting of entactin, heparan sulfate proteoglycans (HSPG), growthfactors and combinations thereof.
 11. The cell culturing substrate ofclaim 1, wherein the aqueous solvent is selected from the groupconsisting of a buffer, a cell culture media and combinations thereof.12. A method of culturing stem cells comprising: providing a cellculturing substrate according to claim 1; applying a suspension of stemcells to said cell culturing substrate; incubating said suspension ofstem cells on said cell culturing substrate at 5% CO₂ in humidified airat 37° C.; and permitting said stem cells to attach to said cellculturing substrate of the cells, wherein the attached cells remain in apredominately undifferentiated state.
 13. The method of claim 12,further comprising introducing a cell culture medium to said cellculturing substrate prior to application of said suspension ofeukaryotic stem cells.
 14. The method of claim 12, wherein said cellculture medium comprises growth media.
 15. The cell culturing substrateof claim 12, wherein said stem cells are eukaryotic stem cells.
 16. Thecell culturing substrate of claim 12, wherein said stem cells areembryonic stem cells.
 17. The cell culturing substrate of claim 12,wherein said stem cells are human embryonic stem cells.
 18. The cellculturing substrate of claim 12, wherein said stem cells are humanmesenchymal stem cells.
 19. The cell culturing substrate of claim 12,wherein said stem cells are human neuronal stem cells.